Microbiota-dependent indole production stimulates the development of collagen-induced arthritis in mice

Altered tryptophan catabolism has been identified in inflammatory diseases like rheumatoid arthritis (RA) and spondyloarthritis (SpA), but the causal mechanisms linking tryptophan metabolites to disease are unknown. Using the collagen-induced arthritis (CIA) model, we identified alterations in tryptophan metabolism, and specifically indole, that correlated with disease. We demonstrated that both bacteria and dietary tryptophan were required for disease and that indole supplementation was sufficient to induce disease in their absence. When mice with CIA on a low-tryptophan diet were supplemented with indole, we observed significant increases in serum IL-6, TNF, and IL-1β; splenic RORγt+CD4+ T cells and ex vivo collagen-stimulated IL-17 production; and a pattern of anti-collagen antibody isotype switching and glycosylation that corresponded with increased complement fixation. IL-23 neutralization reduced disease severity in indole-induced CIA. Finally, exposure of human colonic lymphocytes to indole increased the expression of genes involved in IL-17 signaling and plasma cell activation. Altogether, we propose a mechanism by which intestinal dysbiosis during inflammatory arthritis results in altered tryptophan catabolism, leading to indole stimulation of arthritis development. Blockade of indole generation may present a unique therapeutic pathway for RA and SpA.

SINA (1.3.0-r23838)(11) using the 418,497 bacterial sequences in Silva 115NR99 (12) as reference configured to yield the Silva taxonomy; taxonomic assignments used the lowest-common-ancestor approach with default SINA settings.Closed-reference, operational taxonomic units were produced by binning sequences with identical Silva/SINA LCA assignments.Taxa with >0.01%abundance in any sample and observed in >5% of the samples were included in further analyses.All samples had a Good's coverage index >99%, indicating excellent depth of sequencing coverage.
The software packages R (v3.6.3)(13) and Explicet (v2.10.5)(14) were used to analyze and visualize data.Alpha-diversity indices (i.e., Chao1, Shannon H, Shannon H/Hmax) were evaluated between groups by ANOVA.Differences in overall microbiota composition (i.e., beta-diversity) were assessed through permutational ANOVA (PERMANOVA) with the Aitchison dissimilarity index applied to sequence count data (15,16).Principal coordinates analysis (PCoA) was carried out using Aitchison dissimilarities and the wcmdscale function in the vegan R package.(16) Individual taxa differing between treatment groups were identified using the ANOVA-like differential expression (ALDEx2) R package (17,18).The distribution of taxa in each sequence library was estimated through 1000 Dirichlet Monte Carlo re-samplings of sequence count data.To account for the compositional nature of microbiome sequence data, datasets were then subjected to a center log-ratio transformation with all features used as the denominator.P-values were adjusted for multiple comparisons using the false discovery rate method (19).Effect size plots are derived from the outputs of ALDEx2 and represent the median effect sizes, calculated as the median between-group difference in CLR values between groups divided by the largest within-group difference in CLR values (17,18).
Metabolomics.Cecal tip (tissue and contents, 30-100 mg) were harvested at day 35, flash-frozen, and stored at -80 o C. Metabolomic analyses were performed via one of three methods as follows: HPLC: Metabolites were extracted as previously described (20) with minor variations.Briefly, cecal tissue and contents were reconstituted in 200µl of HPLC-grade 80% methanol, sonicated for 3 x 3 second pulses (BioLogics Inc., 150 V/T Ultrasonic Homogenizer, power output ∼20%), and then centrifuged at 12,000g for 5 minutes.The supernatant was saved and the extraction was repeated for a total of three rounds, producing a total of 600µl of extract.Samples were filtered through 5kDa spin columns (Amicon) and metabolites analyzed by HPLC.
Analyses were performed on an Agilent Technologies 1260 Infinity HPLC using a Sepax Br-C18 column (120 Å, 4.6 x 250 mm).Mobile phase A, HPLC-grade water pH 7.0; mobile phase B, HPLC-grade acetonitrile; column temperature at 30 o C and flow rate of 1 ml/min.Chromatographic separation of the metabolites was performed using a gradient of 10% to 90% B in 30 min followed by washing and equilibration periods at the end of each run.The indole derivatives were detected by absorption at 280 nm and their absorbance spectra and retention times were confirmed by co-injection with authentic standards.Area under the curve (AUC) was calculated for each metabolite and normalized to starting sample weight.

HPLC-MS:
Indole derivatives were quantified in mouse cecal samples using reversed-phase high-performance liquid chromatography with electrochemical coulometric array detection (EC-HPLC; CoulArray, Thermo Scientific, Waltham, MA).Cecal samples were extracted in 80% methanol and protein precipitate was removed by centrifugation at 15,000 x g.Separation was achieved using an Acclaim Polar Advantage II C18 column (Thermo, Waltham, MA) at a flow rate of 1 ml/min on a gradient of 10% to 55% acetonitrile in 50 mM sodium phosphate buffer, pH=3, containing 0.42 mM octanesulphonic acid as an ion-pairing agent.Calibration curves were composed by performing linear regression analysis of the peak area versus the analyte concentration.The data were quantified using the peak area in comparison to standards.LC-MS/MS: cecal metabolites were analyzed by LC-MS/MS as described previously (21).

Detection of CII-specific antibodies.
Type II collagen-specific antibodies were detected in mouse serum at CIA day 35 using previously published methods (22).Briefly, 96-well plates (Nunc MaxiSorp) were coated overnight at 4C with 5µg/ml ELISA-grade bovine CII (Chondrex), washed 3x with PBS + 0.05% Tween-20, and blocked for 4hr at 4C with 1% BSA in PBS.Serum samples were added to the wells at a dilution of 1:40,000 and incubated overnight at 4C with rocking.The plates were then washed, and horseradish peroxidase (HRP)conjugated goat anti-mouse IgG, IgG1, IgG2a, or IgG2b (SouthernBiotech, Supplemental Table 4) were added to the wells at a dilution of 1:10,000 for 4 hours at 4C.Following washing, 1X TMB ELISA substrate solution (BD Biosciences) was added to the wells and the plates were developed at room temperature.The reaction was stopped with 2N H 2SO4 and read at 450nm and 570nm wavelengths.Pooled serum from mice with severe CIA was used to generate a standard curve in which the top standard was diluted 1:1000 (1unit/ml), followed by 2fold serial dilutions.

C3 activation ELISA.
Complement binding to CII-specific antibodies was assessed using previously published methods (1).Ninety-six-well plates were coated overnight at 4°C with 5 μg/ml ELISA-grade mouse CII.The ELISA plates were then washed three times with 0.1% BSA + 0.05% Tween-20 in 1X PBS and blocked with 1% BSA in 1× PBS for 4 hours at 4°C.Serum samples were diluted 1:10,000 in 1× PBS.Samples were added to wells and incubated overnight at 4°C.The next day, wells were washed 5 times with 1× PBS + 0.05% Tween-20, then incubated with 15% normal mouse serum diluted in Dulbecco's PBS + 0.9 mM CaCl2 + 0.5 mM MgCl2 for 30 minutes at 37°C.The plates were then washed 5 times with 1× PBS and incubated with HRP-conjugated goat IgG to mouse C3 (Cappel/MP Biomedicals, Supplemental Table 4) in 1:2,500 dilution for 1 hour at room temperature with rocker shaking.The plates were washed 5 times with 1× PBS and developed with 1× TMB ELISA substrate solution for 10 minutes.The reaction was then stopped with H2SO4 and read at 450 nm.
Glycosylation studies.Serum total IgG was purified by using Pierce Protein G Agarose (ThermoFisher Scientific) following the manufacturer's instructions.CII-specific antibodies were purified from serum as previously described (23) by coupling bovine CII (Chondrex) to CNBr-activated Sepharose 4B beads.To concentrate the eluted total IgGs, 3-kd Ultra-0.5 ml centrifugal filter units (Amicon) were used.Total N-linked glycan was released from glycoproteins using PNGase F (New England Biolabs) according to the manufacturer's instructions.Deglycosylation reactions were carried out at 37°C overnight to ensure effective release of glycans.
Glycans were purified from the reaction using GlykoClean G Cartridges (Prozyme), dried, and fluorescence labeled with 2-aminobenzamide (Sigma-Aldrich).Labeled glycans were cleaned with GlykoClean S-plus Cartridges (Prozyme), dried, and subjected to high-performance liquid chromatography analysis.Glycan samples were dissolved in 25% 100 mM ammonium formate (pH 4.5) and 75% acetonitrile then separated using an Agilent 1260 Infinity Quaternary LC system outfitted with a 2.1 × 150 mm AdvanceBio Glycan Mapping column with 2.7 μm superficially porous particles and a fluorescence detector.Resulting peaks were analyzed in OpenLAB software (Agilent) and assigned glycoforms by comparing peaks of commercially available human IgG N-linked glycan library.Flow analysis.Tissues were harvested from mice with CIA at day 21 or 35, homogenized in RPMI media, and passed through a 70 micron cell strainer.Red blood cells were lysed using Red Blood Cell Lysis buffer (eBioscience), and resuspended in FACS buffer (5% FBS in PBS) for surface staining.For intracellular staining, cells were fixed and permeabilized using the FoxP3/Transcription Factor Staining Buffer Kit (Tonbo).All antibodies and clones used are listed in Supplemental Table 3. Flow cytometric analysis was performed at the Barbara Davis Center BioResource Service Center, and analysis was performed using FlowJov10 software.
Splenocyte re-stimulation.Splenocytes were harvested as described above and re-stimulated with UVcrosslinked bovine Type II Collagen at a final concentration of 500ug/ml for 72 hours.Alternatively, 5x10 5   splenocytes were stimulated with 2µl pre-washed CD3/CD28 Dynabeads.Supernatants were stored at -20C and were analyzed by multiplex immunoassay (MSD) as described above.
Histopathology.Mouse paws were removed at mid-limb and fixed in 10% paraformaldehyde.The bones were decalcified in 10% formic acid for 1 week and then embedded in paraffin.Sections of 5μm were cut from paraffin embedded tissues and stained with H&E.Pathology was assessed in a blinded manner for inflammation, pannus formation, and bone erosion.Pannus scoring criteria: 0 = no areas affected; 0.5=Very minimal, marginal zone only, less than 1% of area at risk affected; 1=Minimal infiltration of pannus in cartilage and subchondral bone, marginal zones mainly.Approximately 1-10% of area at risk affected; 2=Mild infiltration with marginal zone destruction of hard tissue in affected joints, 11-25% of area at risk affected; 3=Moderate infiltration with moderate hard tissue destruction in affected joints, 26-50% of area at risk affected; 4=Marked infiltration with marked destruction of joint architecture, affecting most joints, 51-75% of area at risk affected; 5=Severe infiltration associated with total or near total destruction of joint architecture, affects all joints, greater than 75% of area at risk affected.Bone resorption scoring criteria: 0.5=Very minimal resorption affects only marginal zones; 1=Minimal approximately 1-10% of area at risk of subchondral bone affected; 2=Mild, more numerous areas of resorption, approximately 11-25% of total area at risk of subchondral bone affected; 3=Moderate, obvious resorption of subchondral bone resulting in approximately 26-50% of area at risk of subchondral bone affected; 4=Marked, very obvious resorption of subchondral bone resulting in approximately 51-75% of area at risk of subchondral bone affected; 5=Severe, distortion of entire joint due to destruction approximately 76-100% of area at risk of subchondral bone affected.Inflammation scoring criteria: 0=no inflammatory infiltrate; 1=mild cellular infiltrate into joint and synovium; 2=enhanced cellular infiltrates, increased cell density throughout the joints, some joints affected; 3=maximal inflammation, high cell density, all joints affected.Complement C3 Immunohistochemistry. Paraffin-embedded tissue slides were assessed for C3 complement deposition in the joints as described previously (24) using Goat Anti-Mouse Complement C3 (MP Biomedical, Supplemental Table 4).Complement deposition in each of the four paws was scored by a blinded observer from 0 to 3 (0=no staining, 1=mild staining, 2=moderate staining, 3=intense staining), and the average score across the four paws was plotted.

Collection and Isolation of Human LPMC. Colon tissue samples (N=5) were procured from the Program for
Individuals with an Elevated Risk of Spondyloarthritis (PIERS) Registry.Healthy tissue was obtained from patients undergoing bowel surgery and would otherwise be discarded.These patients had no existing rheumatic disease or a history of Inflammatory Bowel Disease, HIV-1 infection, current treatment with immunosuppressive drugs, or recent chemotherapy (within 8 weeks).All patients undergoing surgery consented to the use of discarded tissue for research purposes.Protected patient information was de-identified to the laboratory investigators.
Briefly, tissue specimens were trimmed of muscle and fat and treated with DL-Dithiothreitol (DTT; 1.67mM; Sigma-Aldrich) to remove additional mucus.The epithelial layer was subsequently removed with 1mM EDTA (Sigma-Aldrich) and the remaining tissue treated with collagenase D (0.5mg/ml, Roche Diagnostics).All released LPMCs were cryopreserved and stored in liquid nitrogen.
Cryopreserved LPMCs were thawed and stimulated at 37C with 1mM indole for 4 hours.CD3+ T cells and CD19+ B cells were flow sorted using a FACSAriaIII cell sorter.Total RNA was extracted using an RNeasy kit (Qiagen) according to the manufacturer's protocol.Libraries were constructed as previously described using the Next Ultra II directional RNA library prep kit with rRNA depletion (28).Bulk RNA sequencing was performed on an Illumina MiSeq platform at the University of Colorado Genomics core.RNA-Sequencing workflow was implemented through the Bioconductor differential expression pipeline (29,30).Salmon (31) was used for transcript quantification with GC bias correction, using a human transcript reference index from GENCODE (release 38) and no decoy sequences (32).Tximeta (33) was used for importing transcripts which were then analyzed for differential expression with DESeq2 (34).Ingenuity Pathway Analysis as used to identify differentially expressed pathways.Differentially expressed pathways were defined as those with a p-value <0.05 and a z-score >2.